PROJECT SUMMARY/ABSTRACT The motivation for these studies is the need to gain an understanding of the fundamental biophysical properties of the skeletal voltage-gated L-type Calcium channel CaV1.1. While ion conduction is a critical feature (and often the only duty) of the vast majority of ion channels, CaV1.1 is somewhat unique: the activation of its voltage sensors opens its pore, but Ca2+ entry is not required to trigger muscle contraction. Instead, the conformational changes of Cav1.1 voltage sensors directly gate Ryanodine receptors (RyR1) via a physical coupling between these two channels, to trigger the release of sarcoplasmic reticulum Ca2+. In this context, the four voltage-sensing elements of Cav1.1 are indeed the voltage sensors of RyR1 channels. As the possibility to express Cav1.1 channel in oocytes has recently become feasible thanks to the discovery of an essential adaptor protein (Stac3), the Olcese laboratory is in a privileged position to directly address the mechanism of voltage regulation in this protein, with a unique capability (to date) to implement the cutting-edge voltage clamp fluorometry approach to CaV channels. During the next five years, using electrophysiological, optical and computational techniques, the investigators will delineate the basis of voltage dependence in CaV1.1 channels, in both adult and embryonic splice variants. They will interrogate how this voltage dependence is modulated by the participation of auxiliary subunits (?, ?2?, and ?) in the CaV1.1 macromolecular complex. They will determine which of the four homologous, but non-identical CaV1.1 Voltage Sensing Domains confer voltage sensitivity to RyR1-mediated Ca release. Finally, the investigators will address the molecular mechanism of a malignant-hyperthermia-causing mutation that specifically affects the voltage-sensing apparatus of CaV1.1. The knowledge gathered by is critical to understand fundamental aspects of muscle physiology and the voltage-dependent control of contraction.